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Instrument-free Point-of-care Device for Detection of Pathogens and Antimicrobial resistance

Primary Information

Domain

Healthcare

Project No.

7075

Sanction and Project Initiation

Sanction No: 7075

Sanction Date: 18/03/2017

Project Initiation date: 18/03/2017

Project Duration: 36

Partner Ministry/Agency/Industry

Ministry of Health and Family Welfare SRL Diagnostics Ltd, Mumbai

 

Role of partner:The industry participation is of SRL Diagnostics Ltd., one of the largest Diagnostics Company in India, with access to a wide range of clinical samples through an efficient network of labs and collection points. This will provide ease of access to samples and will provide feedback from requirements from the industry. The collaboration will help in device validation as the results are compared to clinical gold standards of microscopy and culture.

 

Support from partner:Regular meetings with the team at SRL Diagnostics Ltd, Mumbai have been conducted right from the development of the concept to the present day. The devices are being developed with continuous inputs from experts working with the clinical and industry partners.Thus, industry inputs from the experts at SRL Diagnostics Ltd has been initiated at the very beginning of the study to ensure a clinically validated point of care device which is relevant in the Indian scenario. The continuous provision of clinical specimens and allowing usage of facilities to analyse the devices being developed is a vital support from our partners.

Principal Investigator

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Prof Rinti BanerjeePrimary
Indian Institute of Technology Bombay

Host Institute

Co-PIs

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Dr Gita Nataraj
GS Medical College and KEM Hospital, Mumbai

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Dr B R Das
SRL Diagnostics Ltd

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Dr Rishi Adhikary
Indian Institute of Technology Bombay

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Dr Biji Balakrishnan
Indian Institute of Technology Bombay

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Dr. Sucheta P. Dandekar
Seth G. S. Medical College and KEM Hospital

 

Scope and Objectives

Infections are important clinical problems in India. Antibiotic prescription in any infection requires microbial culture and drug sensitivity analysis. However, because of the cost, time, expertise and infrastructure involved in culture and sensitivity, often the antibiotic prescription is based on the sheer experience of the physician without taking into consideration the underlying pathogenic organism. Due to this non evidence based prescription of antibiotics, there is gradual development of drug-resistant organisms. In such a scenario, irrational treatments for infections would only complicate matters resulting in increased drug resistance and costs of treatment. This is leading to increase in the cost of therapy due to the higher price of the second line of therapy and also the indirect costs involved in multiple visits to the health facility. With the advances in nanotechnology and new age smart materials it is now possible to modify conventional microbiological tests are lengthy, expensive and require skilled manpower which limit accessibility and affordability in resource limited settings. We intend to create affordable POCDs, firstly to determine the presence of bacteria in a clinical sample, device to determine antimicrobial resistance using drug loaded nanoparticles incorporated for faster determination of the antimicrobial to be prescribed. Finally a device to process complex samples like blood. The project has three inter-dependant arms as mentioned. The development of these devices would be instrumental in implementation of the National Action Plan, 2017 to combat antimicrobial resistance by helping in surveillance as well as by promoting rational use of antibiotics. Objectives Develop a point of care screening device for detection of bacterial or non bacterial infections Develop a point of care screening device for detection of drug sensitivity of common antibiotics Develop a sample processing unit for complex samples

Deliverables

At the end of Grant period following deliverables will be developed Point of care device to determine presence of bacteria in samples Point of care device with series of tests to identify bacteria and its Antibiotic susceptibility Sample preparation /concentration device for complex samples like blood Three prototypes have ben developed

 

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Scientific Output

Test for presence of bacteria in sample Strategy I This test is based on entrapment of bacteria on 0.2 micron filter substrate followed by suitable staining and decolourization steps and observing colour of the membrane. It stains blue in case of presence of bacteria and remains white for a non-bacterial sample. The test was assessed using a wide range of Gram positive and Gram negative cultures Preliminary results indicated a clear determination for saline suspensions and spike samples (Urine and serum). This technology was transferred to SRL for validation with clinical specimens, who suggested changes for easy interpretation of results through modifications or through an alternate technology. Strategy II The alternate technology is based on use of dye entrapped bio-polymeric nanoparticles. The positively charged nanoparticles conjugated with specific bacterial binding proteins that form clumps with bacteria in a culture positive sample visible to naked eye. While the culture negative sample remains coloured suspension indicating absence of bacteria. With 100 percent specificity and sensitivity observed in saline suspensions and spiked urine samples. So far the technology is being evaluated with 50 clinical samples. Based on the results the test is being finalised with minor modifications to improve the specificity for bacterial cells and rule out any false positive due to presence of eukaryotic cells in the sample. Test for identification of bacteria Additional deliverables were achieved in terms of identification of individual bacteria without any culture requirements. Peroxide based hydrogels (Modified Catalase Test) were developed, that are stable for more than 60 days and validated by SRL. The report suggested the test was easy to perform and has clear interpretation of results. The test can identify both pyuria in clinical samples and bacteriuria (S. aureus, P. aeruginosa, and P. mirabilis). It can be used as screening test for preliminary identification of infections in cases of urinary tract infections and to determine presence of specific bacteria from cell free samples. With 100 percent sensitivity and specificity in saline suspension and spiked urine samples. This test has been evaluated with 50 clinical samples of urine, showing 100 percent sensitivity and 97percent specificity. Modified Oxidase test, specifically identifies P. aeruginosa in a sample. Instant development of blue colouration in the gel for a sample containing P. aeruginosa, makes the test highly specific for identification of the bacterium. The test is under clinical validation. Modified gelatinase test with silver nanoparticles has been developed, simple colour change based reaction identifies presence of S. aureus and E. faecalis in cell free samples sample. Incorporation of these tests into a single test format is being attempted for detection of various infections. Test for Antibiotic sensitivity Test for determining presence of Extended Spectrum Beta Lactamase ESBL enzyme and assessed with ATCC cultures of ESBL and MRSA along with other susceptible strains. Validated by SRL and concluded that the limit of detection needs an improvement to allow detection of clinically relevant concentrations of bacteria. Sample processing unit Various biopolymeric smart materials are being explored for development of a prototype filtration device to separate bacteria from other components of complex samples like blood.

 

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Results and outcome till date

Point of care tests validated in spiked clinical samples is being periodically tested by SRL Diagnostics Ltd. This is being done with clinical specimens like urine, ascitic fluid, pleural fluid and bronchoalveolar lavage. Conclusions from the test are being incorporated to improve the devices for respective tests. The test for bacterial or non bacterial Clinical validation with clinical samples The peroxide based test with 100 percent sensitivity is being used as a screening test for pyuria or bacteriuria Cell free samples. The device remained stable for more than 60 days since the date of preparation and it was still able to identify presence of WBCs or bacteria like P. aeruginosa in clinical samples. Further improvements in the design of the test are being done to incorporate it into various clinical relevant bacterial POC kits. The device for penicillin resistance ESBL identification needs minor improvements for limit of detection based on reports from SRL. Similar devices for MRSA based on internationally approved CLSI guidelines and cephalosporins resistance are being developed and assessed with standard ATCC strains and shortly will be transferred to SRL for clinical tests.

 

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Societal benefit and impact anticipated

While most emerging technologies like molecular techniques and other hi-tech instruments with biochemistry analysis are concentrating on accurate identification of bacteria. Most of these techniques require purified culture obtained after 24-48 hrs. Such technologies are often expensive, require sophisticated laboratories, trained personnel while the reports are generally obtained after 48 hours of patient visit. This is a major hindrance in immediate and effective treatment of the patients, especially in resource limited settings. Thus, there is an utmost requirement of technology for instant screening of samples to determine the causative infectious agent and its antimicrobial susceptibility. Therefore, the POCDs developed will be rapid, affordable, instrument free, will not require any power supply, will require minimal training for administration by a health care worker and will act as an effective screening tool. Thus, reducing the overall time, energy and cost of clinical analysis. With these advantages POCDs developed will revolutionise the diagnostic scheme and benefit the society at large. In the current scenario where drug resistance is a major concern for the entire human race, clinicians and health care workers can be of great help in tackling this by incorporating such POCDs in their routine analysis and follow a judicious use of antibiotics for patients across all economic backgrounds. The POCDs can be easily administered and incorporated into the Antibiotic Stewardship Program of a health care facility. The POCD will be able to effectively determine the causative pathogen for various infections. This would hugely impact the antibiotic prescribed. It would be based on the causative organism rather than a guesswork. So, the hospitals and primary health centres will be the potential users of this device. Ultimately, the end users will be the patients with infectious disease. This would gradually reduce drug resistance and improve the care for infections in remote areas of developing nations.

Next steps

The most immediate steps planned are clinical validation of the devices developed to ensure sensitivity and specificity of at least above 80 percent. Other related parameters are interference studies, stability, feasibility and packaging of devices. Followed by the development of filtration devices for complex samples, and cocktail media comprising of lytic agents against eukaryotic cells and enrichment components of bacterial cells. At the end of the tenure POCDs with following Target product profile Instrument free, no microscope No culture or pure colonies required 80 percent sensitivity, 80 percent specificity Sample requirement less than 500 micro-litres Time of read out less than 3 hours Processing of complex samples Stable over 1 year

Publications and reports

Nil

Patents

Patent filed Rinti Banerjee, Adhikary Rishi, Baby Jason, Agarwal Ratika, A Substrate for Entrapment and Detection of Bacteria, Process for its Preparation And A Point of Care Device Having the Substrate 47078 2017-MUM

Scholars and Project Staff

i. Dr. Kapil Punjabi Research Associate 12 months ii. Ms. Diti Shah Junior Research Fellow 12 months iii. Ms. Ahana Chatterjee Mtech Biomed 12 months iv. Ms. Aishani Patnaik Mtech Biomed 12 months v. Ms. Vaishnavi Samant Intern 6 months vi. Ms. Michelle Dias Interns 4 months vii. Ms. Sameeksha Bhaye 4 months viii. Ms. Prachi Bendale 6 months ix. Ms. Neha Singh 4 months

Challenges faced

No major difficulties

Other information

Three prototype devices have been developed

Financial Information

  • Total sanction: Rs. 14997000

  • Amount received: Rs. 5878000

  • Amount utilised for Equipment: Rs. 226270

  • Amount utilised for Manpower: Rs. 1268688

  • Amount utilised for Consumables: Rs. 1166499

  • Amount utilised for Contingency: Rs. 1778544

  • Amount utilised for Travel: Rs. 31009

  • Amount utilised for Other Expenses: 0

  • Amount utilised for Overheads: Rs. 1106283

Equipment and facilities

 

Hyrel Bioprinter obtained for the printing of hydrogel based tests and other point-of-care devices.